During the past three decades immunotherapy has become an important approach for treating human diseases and conditions through the use of regimens designed to modulate immune responses. This is particularly important in pathological conditions where the immune system becomes compromised. Studies conducted in disease models and clinical trials demonstrate that augmenting host defense mechanisms is useful in treatment and prophylaxis against microbial infections, immunodeficiencies, cancer, and autoimmune disorders (1-5). Immune enhancing protocols may also have utility for promoting wound healing. In the process of wound healing, macrophages exhibit a principal role by modulating cellular proliferation and new tissue formation/regeneration. They also function as phagocytes, debridement agents and produce growth factors that influence the angiogenesis stage of wound repair (6).
Most immunostimulants of natural origin are high molecular weight polysaccharides, glycoproteins or complex peptides (1). For example, three fungal polysaccharides derived from Schizophyllum commune (schizophyllan), Lentinus edodes (lentinan) and Coriolus versicolor (krestin) have been clinically used in Japan as biological response modifiers (4). Another polysaccharide, acemannan (isolated from Aloe vera), is licensed by the United States Department of Agriculture for the treatment of fibrosarcoma in dogs and cats (7). There are a few small molecular weight immunostimulants derived from natural products such as the glycosphingolipid KRN-7000 (8). Several immunostimulants of synthetic origin also have been developed that include compounds like isoprinosine and muramyl peptides (2). A number of other immunomodulators of endogenous origin have been developed using recombinant technologies that have gained FDA approval. These agents include colony-stimulating factors, interferons and recombinant proteins (5). However, these compounds often have short half-lives and it is difficult to determine optimal dosage and appropriate combinations.
Although current immunostimulants show promise, there is still a need to develop more potent agents and increase the arsenal of available drugs for immunotherapy. One source of chemically diverse compounds that can be used for drug discovery of immunostimulants is natural products. For centuries natural products have been exploited as therapeutically useful agents, many of which are in clinical use today. Interest in natural products as a means to drug discovery is based on their unparalleled molecular diversity and rich spectrum of biological activities (9).
Since ancient times, microalgae have been used as a nutrient-dense food source. Historical records indicate that microalgae such as Spirulina platensis was consumed by tribes around Lake Chad in Africa and by the Aztecs living near Lake Texcoco in Mexico (10). During the last several decades there has been increasing interest in the commercial production of food-grade microalgae for human consumption and as feed for livestock. Among the various microalgae that have been explored for their commercial potential Spirulina species, Chlorella species and Aphanizomenon flos-aquae (AFA) are three major types that have been successfully produced and are in widespread use. Other food-grade microalgae include Dunaliella salina and Haematococcus pluvialis. 
Both anecdotal reports and recent studies on the consumption of food-grade microalgae have reported enhanced immune function in both animals and humans. Oral administration of Chlorella vulgaris has been correlated with enhanced natural killer cell activity (11) and granulocyte-macrophage progenitor cells (12) in mice infected with Listeria monocytogenes. Dietary Spirulina platensis increases macrophage phagocytic activity in chickens (13) and Spirulina fusiformis exhibits chemopreventive effects in humans (14). Human consumption of AFA has been reported to produce changes in immune cell trafficking and enhanced immune surveillance (15). The active components for all these effects have not been conclusively established.
Chlorella Polysaccharides and Glycoproteins
A number of polysaccharides have been identified from Chlorella species that possess biological activity. In U.S. Pat. No. 4,533,548 an acidic polysaccharide was isolated from Chlorella pyrenoidosa that exhibits antitumor and antiviral activity (16). The glycosyl composition for this polysaccharide was mostly rhamnose, with minor amounts of galactose, arabinose, glucose and glucuronic acid. Another polysaccharide, isolated from marine Chlorella minutissima, reported in U.S. Pat. No. 4,831,020, appears to have tumor growth-inhibiting effects. However, no molecular weight or glycosyl composition was reported (17).
In U.S. Pat. No. 4,786,496, the lipid fraction (glycolipid portion) of marine Chlorella species displayed antitumor properties (18). Several glycoproteins have also been isolated from Chlorella species. For example, U.S. Pat. No. 4,822,612 reported a 45,000 dalton glycoprotein that has anticancer effects (19). Various other glycoproteins (20-23) and glyceroglycolipids (24) that may have immunopotentiating and antitumor properties also have been reported in the scientific literature. None of these compounds are polysaccharides.
Spirulina Polysaccharides
Several different types of polysaccharides that exhibit biological activity have been isolated from Spirulina species. For example, the sulfated polysaccharide calcium spirulan inhibits tumor invasion and metastasis (25). Calcium spirulan (molecular weight 74,600 daltons) is composed of rhamnose (52.3%), 3-O-methylrhamnose (32.5%), 2,3-di-O-methylrhamnose (4.4%), 3-O-methylxylose (4.8%), uronic acids (16.5%) and sulfate (26).
U.S. Pat. No. 5,585,365 discloses that an antiviral polysaccharide with a molecular weight between 250,000 and 300,000 daltons was isolated from Spirulina species using hot water extraction (27). This polysaccharide is composed of rhamnose, glucose, fructose, ribose, galactose, xylose, mannose, glucuronic acid and galacturonic acid. A number of other low molecular weight polysaccharides that range between 12,600 and 60,000 daltons recently have been isolated from Spirulina species (28-30).
Previous Work by the Inventors
The present inventors have characterized novel polysaccharide preparations from the microalgae Spirulina platensis, Chlorella pyrenoidosa and Aphanizomenon flos-aquae (31). These are high molecular weight preparations that contain polysaccharides with methylated and acetylated sugars and therefore are extractable to some extent with water and also under more non polar conditions such as with aqueous alcohol.
The present inventors have also recently described a previously unrecognized class of immune stimulants and methods for their quantitative isolation from plant material (32). This class of compounds was identified as a melanin and it retains its ability to activate monocytes after isolation. It represents a major portion of the immunostimulatory activity of these botanicals.
In the present invention the inventors have applied this isolation method to quantitatively extract melanin from the following food-grade microalgae and algae: Spirulina platensis, Chlorella pyrenoidosa, Aphanizomenon flos-aquae, Haematococcus pluvialis and Fucus vesiculosis (kelp). There has not been a report of the existence of immunostimulatory melanin within these microalgae and algae.
Melanins
Melanins are complex pigment polymers that occur throughout nature. In mammalian tissue the two main types of melanin are eumelanins (black colored material that is insoluble in most solvents) and pheomelanins (yellow or reddish brown, alkali-soluble pigments). Biosynthetically, these animal melanins are derived from tyrosine oxidation by tyrosinase (coupled with thiols such as glutathione or cysteine for pheomelanins). In microorganisms, such as fungi, most melanins are derived from 1,8-dihydroxynaphthalene and contribute to virulence and modification of host immune responses (33). Although a few reports have been published on plant melanins, definitive structural data is often lacking. Some of the biological effects attributed to melanins include direct acting antiviral activity (34) and the more commonly known photoprotective/redox properties (35). The inventors have found only one report of a “melanin-like” material exhibiting immunostimulatory activity. This material was isolated from black tea leaves and when orally administered to mice, enhanced the antibody response of spleen cells to sheep red blood cells in as little as two days (36).
The insolubility of melanins in common solvents has been a major obstacle in both initial extraction as well as purification schemes. Two general isolation approaches have been developed. The first approach isolates melanin by removal of all other substances from the initial material. This elimination process typically involves harsh chemical treatments with strong acids and base. The major problems with this approach is that the melanins remain contaminated with other classes of compounds and the harsh isolation conditions leads to the destruction of native melanin structure. The second approach extracts alkali soluble melanins with either strong base at high temperatures (for example 0.5 to 3 M sodium hydroxide) or weak base (2% ammonium hydroxide) at room temperature. The immune stimulatory properties of some melanins may have been missed due to harsh treatment with base since the present inventors have found that treatment of melanin with 0.5 M sodium hydroxide completely destroys its ability to activate monocytes (32). However, the inventors have found that melanin can be qualitatively extracted using weak base and under these conditions it retains its ability to activate monocytes. Since it can be solubilized in weak base it can be subjected to chromatographic analysis. The sensitivity to strong base in addition to melanins limited solubility in commonly used solvents may explain why previous investigators did not detect this potent immunostimulatory compound in botanical material. The present inventors have developed an efficient and quantitative isolation procedure based on initial extraction with aqueous phenol that results in melanin preparations from plant material of high purity while maintaining its immunological activity (32).
Monocyte/Macrophage Activation System
One way to determine immunostimulatory activity is to use a biological assay involving macrophages. Monocytes/macrophages are found in practically every tissue of the body where they are critical in coordinating immune responses and numerous biological processes (37). They play a major role in phagocytosis, immune surveillance, wound healing, killing of microbes and tumor cells, and antigen presentation to T lymphocytes (38). In cancer, macrophages mediate tumor cytotoxicity functions through the production of cytokines and other immune factors (39). In order for macrophages to play a major role in adaptive and innate immunity they must respond effectively to environmental agents by first becoming activated (40). Macrophage activation is mediated by proinflammatory transcription factors such as nuclear factor kappa B (NF-kappa B). Such transcription factors then control and modulate the activation/repression of an array of genes that mediate a variety of immune responses.
In unstimulated macrophages, NF-kappa B exists as inactive heterodimers sequestered by inhibitory-kappa B (I-kappa B) proteins within the cytosol. Agents that cause I-kappa B proteins to dissociate and degrade allow for the translocation of NF-kappa B dimers to the nucleus where they can activate transcription of downstream genes (41). Target genes regulated by NF-kappa B include proinflammatory cytokines, chemokines, inflammatory enzymes, adhesion molecules and receptors (42).
In this invention a transcription factor based assay for NF-kappa B in human monocytes was used to guide extraction, isolation, characterization and development of immunostimulatory melanin preparations from food-grade microalgae/algae. The melanins of the present invention represent a novel class of immune stimulants.